Bilirubin is a degradation product of hemoglobin. It has been estimated that approximately 200-230 mg of bilirubin and its derivatives are formed each day in a normal human adult by the degradation of hemoglobin within the liver, spleen, and bone marrow. Bilirubin has a natural yellow-orange color.
In human body fluids such as bile and serum, bilirubin exists in chiefly two different forms, these forms commonly being referred to in the clinical literature as conjugated bilirubin, B.sub.c, and unconjugated bilirubin, B.sub.u. Therefore, the total bilirubin content, B.sub.T, of any individual body fluid commonly represents the sum of the B.sub.u and B.sub.c components in that fluid.
The diagnostic significance of bilirubin is well established. For example, an excessive amount of bilirubin within the human body, referred to as jaundice, is recognized as evidence of a variety of disease conditions, particularly diseases of the liver.
The literature on various bilirubin assay techniques and methodology is voluminous. Direct, spectrophotometric analysis represents one of the more popular methods for determination of bilirubin owing to its simplicity. Direct, spectrophotometric assays determine the presence and/or concentration of a substance in a liquid by detecting the absorption spectrum of the liquid in a spectral region in which the substance exhibits inherent molar absorptivity.
One particularly useful direct, spectrophotometric assay recently developed for bilirubin is described in Wu et al, U.S. Pat. No. 4,069,017 issued Jan. 17, 1978. This patent describes a colorimetric bilirubin assay using an interactive mordant for bilirubin. The mordanted bilirubin exhibits an enhanced absorption spectrum, thereby increasing bilirubin assay sensitivity and facilitating quantitative "dry chemistry", as well as "wet chemistry", bilirubin assays.
The problem of providing increased assay sensitivity and maintaining relatively low assay error has been a persistent problem associated with direct, spectrophoto-metric bilirubin assays. This problem has become increasingly troublesome as more stringent precision and accuracy demands are imposed, although some progress has been made. For example, hemoglobin spectral interference with bilirubin assays has been identified. Such hemoglobin interference has been reduced by analyzing the absorption spectrum of a liquid sample at several different wavelengths, one or more being specific to hemoglobin, so that bilirubin spectral absorption readings can effectively be corrected for hemoglobin interference. In addition, as noted above, the use of interactive mordants for bilirubin has enhanced the molar absorptivity of bilirubin so that direct, spectrophotometric bilirubin assays can effectively be made on a quantitative basis, even in "dry chemistry" assays which typically require detectable species exhibiting high molar absorptivities.
Notwithstanding the foregoing efforts, the problem of direct, spectrophotometric bilirubin assay error has persisted, particularly for total bilirubin, B.sub.T, assays conducted on aqueous samples containing a substantial amount of B.sub.c. The present inventor has recently conducted extensive investigation of direct, spectrophotometric B.sub.T assays focusing particularly on the individual B.sub.u and B.sub.c components of a B.sub.T assay. This research has identified important B.sub.c and B.sub.u characteristics heretofore unknown or at least unappreciated. For example, although the molecular structure and molecular weight of B.sub.u is well known, the molecular structure and molecular weight of B.sub.c, owing to its instability, has only recently been elucidated by the present inventor as set forth in his co-pending patent application Ser. No. 56,584 filed July 11, 1979 and in the paper entitled "Human Conjugated Bilirubin-Isolation, Biosynthesis and Molecular Characterization by Direct, Spectroscopic Analysis", T. W. Wu et al, presented at the American Association for Clinical Chemistry 31st Annual Meeting in New Orleans, La., July 15-20, 1979. An abstract of this paper appears in Clinical Chemistry, Vol. 25, No. 6, page 1137, June, 1979. Thus, the molecular weight of a conjugated form of bilirubin, B.sub.c, has been determined to be 918.2 and its molecular structure has been determined to be: ##STR1## where R.sub.1 =glucuronic acid and R.sub.2 =glucuronolactone or R.sub.1 =glucuronolactone and R.sub.2 =glucuronic acid
In addition, the present inventor has recently developed a new technique for extracting highly purified B.sub.c from body fluids, such as bile and serum, as described in his co-pending patent application U.S. Ser. No. 56,585 filed July 11, 1979.
Based on his study of highly purified B.sub.c and B.sub.u, the present inventor has found that B.sub.u and B.sub.c have quite distinct, although overlapping, absorption spectra. For example, although both B.sub.c and B.sub.u absorb in the 435 to 460 nm region of the spectrum, their individual absorption characteristics in this spectral region are quite different. This is highly significant because direct, spectrophotometric assays of B.sub.T are made in the 435-460 nm region of the spectrum using calibrators having a bilirubin content comprised solely of B.sub.u. This is based on the assumption that B.sub.c has an absorption spectrum identical to that of B.sub.u as reported by Henry et al, Clinical Chemistry, Principles And Technics, Harper and Row, p. 1071 (1974). Significant assay errors therefore occur when direct, spectrophotometric B.sub.T assays are carried out in the 435-460 nm spectral region on any aqueous sample having a substantial B.sub.c concentration.
The elimination or at least substantial reduction of the foregoing assay error problem associated with direct, spectrophotometric assays of B.sub.T would permit use of this otherwise highly effective, straightforward approach to B.sub.T assays and would therefore represent a highly useful contribution to the art.